On the TRAIL to Pancreas Cancer-Selective Cell Death (Apoptosis) Introduction: Pancreas cancer was the fourth leading cause of cancer-related mortality in the United States in 2008. There has been little progress in the management of this disease and the annual mortality rate remains nearly identical to the annual incidence rate. Novel therapeutic strategies are desperately needed. TNF-related apoptosis-inducing ligand (TRAIL, Apo2L) exhibits potent, selective apoptotic activity against tumors and is currently under clinical investigation. Bioactive TRAIL is a readily inactivated non-covalent homotrimer. We developed a covalent TRAIL trimer (TR3) which is more potent than recombinant TRAIL with improved stability. Furthermore, TR3 was found to be generically extensible in a stoichiometrically controlled manner. We demonstrate cell-specific targeting without loss of TRAIL activity. This proposal will explore the activity of a pancreas cancer-targeted TRAIL Trimer. Specific Aim 1: To determine the efficacy of multi-domain therapeutics comprised of a mesothelin targeting epitope (scFv) and a covalently linked TRAIL trimer (TR3) toward a panel of human pancreatic cancer cell lines in vitro and to elaborate mechanistic details regarding the role of tumor cell tethering in therapeutic activity. This aim focuses on the molecular biology of the scFv-TR3 fusion constructs. The goal is to determine to what extent an additional receptor/ligand interaction enhances the biologic activity of TR3, to determine whether these modifications potentiate TR3-induced apoptosis, and to delineate the mechanisms by which targeted TR3 has increased activity. Specific Aim 2: To determine the clinical feasibility of mesothelin-targeted TR3 therapy against primary human pancreatic tumor biopsies in vitro and in a xenogeneic mouse model of human pancreas cancer in vivo. This aim will establish whether primary cells from freshly resected pancreas tumors are responsive to TRAIL and furthermore will delineate the therapeutic activity of mesothelin- targeted TR3 to eradicate established human pancreatic tumors in a xenogeneic mouse tumor model. The results obtained from this second aim will establish both the patient population that a targeted TRAIL construct could help as well as be the first component of the preclinical safety and efficacy package for an IND using mesothelin-targeted TR3 for pancreas cancer therapy. PUBLIC HEALTH RELEVANCE: The 5-year survival rate of pancreas cancer patients (4%) remains dismal. New therapies are desperately needed and our TR3 platform technology provides an opportunity to combine a potent biologic agent (TRAIL) with targeting moieties to enhance its therapeutic potential. There is evidence in the literature that such a biologic approach to pancreas cancer therapy may be synergistic with standard of care therapies (such as gemcitabine or radiation). Our team of investigators has the experience necessary to bring promising biologic agents into clinical trials, increasing the likelihood and decreasing the time to translate successful animal studies into patients.